JPH03504980A - Treatment of leukocyte dysfunction using GM-CSF - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Treatment of Leukocyte Dysfunction Using GM-C8F associated with trauma, especially burn injuries The present invention relates to treating leukocyte dysfunction by administering an effective amount of GM-CSF.

GM-C3F is available from Burgess and Metcalf. ), Blood, 56:947 (1980), and Metcalf (Mete alf), Blood, 6e: 257 (1986). It is a lymphokine (immune system stimulator) that exhibits a spectrum of immune cell stimulation. GM -C8F increases white blood cell counts in patients with acquired immune deficiency syndrome [ Brandt et al., N. Engl., J. Med., 31st: 869 ( 1988)] and those with chemotherapy-induced myelosuppression [Antman an) et al., New Engl, J. Med, 319:593 (1988) Various colony-stimulating factors have been shown to be effective alone or in combination with erythrobosomes. yetin and/or antiviral drugs and/or interleukin-2 (I L-2) has been shown to be useful in the treatment of patients with AIDS-type diseases. (PCT International Application No. 87103204).

GM-CSF was confirmed to be able to stimulate the proliferation of hematopoietic progenitor cells; It also stimulates numerous functional aspects of mature granulocytes and macrophages. this Their actions include: biologically active molecules, such as prostagranges; Synthesis of protein E [Hancock et al., J. Immunol, 140 : 3021 (1988), and Kurland et al., P. roc.

Nat], Acad, Sci, USA, 76:2326 (1979) Ko: Food Increased cellular activity [Weisbart et al., Nature, 332 :647 (1988), IL-2 receptor [Hancoc k) et al.

J. Immunol, 140:3021 (1988)] and cells on neutrophils. fungal product formylmethionylleucylphenylalanine receptors (these receptors Sebuter induces the production of superoxide anions) [Atkinson (A tk Immunology, Dan 4: 519 (1988 )] and the regulation of enzyme activity. nodes, such as stimulation of guanylate cyclase and adenylate cyclase Inhibition of [Coffey et al., J. Immunol, 140: 2695 (1988)].

In the case of bodily injury, especially burns, white blood cells (white b) .

It is accompanied by dysfunction of WBC), especially monocytes and leukocytes (]eucocytes).

Therefore, if operating normally, it is involved in, for example, phagocytosis and superoxide production. has a sufficient number of WBCs, but due to white blood cell dysfunction, the immune system is malfunctioning. or inhibition occurs. Immune system dysfunction is caused by white blood cells rather than insufficient white blood cell counts. Caused by blood cell dysfunction.

It is understood by those skilled in the art that such leukocyte dysfunction jeopardizes the recovery of patients with physical injuries. That will be recognized. For example, in burn patients, such impairments (or Increase risk of infection. To date, in vivo leukocyte mechanisms in burn patients No significant treatment of disability and associated clinical consequences, such as treatment of infection. Not talked about.

A welcome contribution to the art is that the leukocyte mechanisms associated with bodily injury, particularly burns, It would be a treatment method for disability. Such a contribution is made by the present invention.

The invention is summarized as follows. Surprisingly, accompanying physical injuries, especially burns, The ability of leukocyte dysfunction to be effectively treated by administration of GM-CSF ( discovered unexpectedly. GM-CSF administered to burn patients improves white blood cell function It has been found that this effect causes an increase in leukocyte function. Therefore this Such treatment significantly increases the response to infection in burn patients. GM - Enhancement of leukocyte function in vivo by C5F is due to the mere number of dysfunctional leukocytes ( It should be distinguished from increasing. Leukocyte dysfunction occurs in types of physical injuries other than burns1 The white blood cell dysfunction that accompanies these other types of physical injuries is similar. It is thought that GM-C3F can be used to treat this disease.

Accordingly, in one aspect, the present invention provides a physical injury in mammals, including humans; GM-CS in an amount effective to reduce associated leukocyte dysfunction and enhance leukocyte function. Provided are methods of treatment by administering F.

In other embodiments, the invention provides methods for treating leukocytes in burn injuries in mammals, including humans. functional impairment by administering an amount of GM-C5F effective to enhance leukocyte function. Provide a method of treatment.

The present invention relates to leukemia associated with physical injuries, such as burns, in mammals including humans. Globular dysfunction is reduced by administration of GM-C3F in an amount effective to enhance leukocyte function. Also provided is the use of GM-C3F in the manufacture of medicaments for use in methods of treating do.

The present invention provides treatment for patients with white blood cell dysfunction associated with bodily injury, such as burns. The use of GM-C3F is also provided.

The present invention further provides for the treatment of patients with leukocyte dysfunction associated with bodily injury, such as burns. Provided is a pharmaceutical composition containing GM-C3F, which is used for the treatment of.

Figure 1 shows the increase in monocyte proliferation in burn patients treated with GM-C3F. This is a rough illustration.

Figures 2 and 3 show the oxidative stress of monocytes in burn patients treated with GM-CSF. 3 is a graphical representation of the increase in burst.

Figures 4 and 5 show that FML P in burn patients treated with GM-C8F. (Formylmethionylleucylphenylalanine) alone over a certain period of time Graphically illustrates the absence of significant monocyte oxidative burst stimulation.

Preferably, the mammal treated is a human and the GM-C3F used is human. Probably an allotype.

In particularly preferred embodiments, an effective amount of GM-C3F is administered intravenously, e.g. by injection. or injection, resulting in significant loss of GM-C3F activity (e.g., replacement of GM-C5F). for a sufficient period of time for GM-CSF to enhance leukocyte function without Administer. Generally, the effective amount is about 3 to about 30 micrograms of GM-C3F/body weight. kg/day, which is administered by intravenous infusion over a period of about 30 minutes to about 24 hours. administered. Preferably the effective amount is about 3 to about 15 micrograms/kg body weight. , which is administered by intravenous infusion over a period of about 2 to about 6 hours, but for about 2 to about 4 hours. Time is more preferred, and about 4 hours is most preferred. The amount used is most preferably 3. 10 or 15 micrograms/kg body weight/day. Actual dose is based on patient's weight and tolerance to GM-CSF.

Unless otherwise specified, the term ``bodily injury'' refers to organ systems, musculature, bones, It refers to damage to various tissues of the body, including the vascular system, etc. Damage is a wound harm, such as thermal burns, electrical burns, chemical burns, blunt injuries, etc. auma), such as those resulting from an accident or assault, traumatic amputations, etc. caused by the action of any mechanism or modality sufficient to cause

Unless otherwise specified, the term ``scald'' is used here to describe excessive heat to an individual. It refers to a physiological injury caused by a chemical burn, and is distinguished from electrical burns and chemical burns.

Unless otherwise indicated, the term “leukocyte dysfunction” as used herein refers to white blood cells, For example, the functionality of monocytes is significantly reduced, or their ability to protect humans from recalcitrant infections. This means that their effectiveness is completely lost. Some of the functions of monocytes and granulocytes includes in vitro or in vivo expression of phagocytosis and/or superoxide production. be caught.

Unless otherwise specified, the term "leukocyte function" as used herein refers to phagocytosis and/or or refers to the normal function of white blood cells, e.g. monocytes, in superoxide production .

Unless otherwise specified, the term ``white blood cell'' as used herein is commonly used in the art. Represents a recognized meaning and therefore includes, for example, myeloid, lymphoid and monocytic cells. Contains various cell types classified as leukocytes, including leukocytes.

The present invention provides a method for enhancing leukocyte function of dysfunctional leukocytes in a mammal. , where this impairment is associated with burns or other physical injuries. this method in an effective amount of GM-C5 for a sufficient period of time to enhance leukocyte function. Administer F. In fact, the method of the present invention significantly reduces leukocyte dysfunction, or Reverse it.

Any suitable GM-C8F may be used in the present invention. Phase for GM-C3F Complementary DNA (cDNA) has recently been cloned and sequenced by numerous laboratories. For example, Gough et al., Nature, 309 Near 63 (1 984) (7 us); Lee et al., Proc, Natl, Acad, Sc i.

Cantrell et al., Proc, Natl, Aca d, Sci.

USA, 82:6250 (1985) (human). Furthermore, non-recombinant GM-CS F was purified from various culture supernatants: Burgers et al., Ex. p.

to). There is nucleotide sequence and amino acid sequence heterogeneity between human GM-CSF. Can be seen. For example, a target at position 100 relative to the N-terminal alanine of human GM-CSF. Both leonine and isoleucine are found in GM-CS within the human population. This suggests that there is an allele of F, ie, a polymorphism. Also, various leader assignments sequences may occur at the N-terminal position of the amino acid sequence. These leader sequences are of various lengths and amino acid compositions, which may influence biological activity. In some cases, it may or may not be given. Preferably, in the present invention, the The GM-C6F used is human GM-CSF (hGM-C8F), and the most preferred Kuhari (Lee) et al., Proc, Natl, Acad, Sci, USA, 82 :4360 (1985) and U.S. Patent Application No. 111.886 Specification I F (filed October 23, 1987) It is recombinant human GM-C3F (rhGM-C5F). All the above references Representative GM-C3F suitable for use in the invention (their DNA and amino acid (including sequence), as well as the preparation method and purification method for GM-C5F. Regarding the publication, it is cited here for reference.

According to the present invention, an effective amount of GM-C8F is administered to a mammal. What is the effective amount? This is the amount necessary to enhance the function of impaired leukocytes. Preferably, as described above, The mammal is a human, and preferred GM-C3F is recombinant human GM-C5F (rh GM-C3F). Generally GM-C3F about 3-30 micrograms/body weight kg/day is sufficient to enhance the function of dysfunctional leukocytes in most patients. It is. Preferably about 3 to about 25 micrograms/kg body weight per day is administered. more preferably about 3 to about 15 micrograms/kg body weight, and 3.10 or 1 A dose of 5 micrograms/kg body weight is most preferred. Even more preferred dose is 10 micrograms/kg body weight.

GM-C3F replaces GM-C5F after the blood concentration of GM-CSF rises rapidly. In contrast to rapid administration, in which the blood concentration of GM-C3F decreases rapidly due to It is best to administer GM-C3F so that its effectiveness is maintained over a certain period of time. Extremely effective. Generally intravenous injection and/or from about 30 minutes to about 24 hours Administration by infusion over time is sufficient. This administration preferably takes about 2 to about 6 hours. , more preferably for about 2 to about 4 hours, most preferably for about 4 hours. . GM-C3F can be administered intramuscularly, subcutaneously, or locally by direct administration to the site of an open wound. , transdermally, intranasally (nasal spray), orally (oral spray), and by inhalation. It can also be administered by other means. Therefore, if the effective blood concentration is Any method of administering the effective amount given is contemplated.

GM-CSF, preferably rhGM-C5F, may be in any of a number of conventional dosage forms. For example, for parenteral use (including sterile solutions or suspensions); for topical use. Dosage forms, e.g. creams, ointments, lotions, transdermal devices (e.g. conventional formulations) Reservoir or Matri Sokus, (Sochi type) etc. .

The formulations and pharmaceutical compositions contemplated by the above dosage forms are conventional pharmaceutically acceptable formulations and pharmaceutical compositions. It can be prepared by conventional methods using excipients and additives.

Currently, GM-C3F, preferably recombinant human rhGM-C3F, is available by intravenous route. administered. The liquid formulations to be administered may be reconstituted from lyophilized powders; Furthermore, it may contain a preservative, a buffering agent, a dispersing agent, and the like. Preferably rhGM-C8F may be prepared in any isotonic medium commonly used for intravenous injections, such as preservative-free. Reconstitute using bacterial water. The maximum concentration of rhGM-C8F is preferably 150 Should not exceed 0 micrograms/ml. Administration is by continuous intravenous infusion or It is given by intravenous injection. For continuous infusion, add the daily dose to saline. This solution is then pumped using a mechanical pump or by gravity. Can be injected.

The following examples are for illustrative purposes only and are not intended to limit the invention in any way. It should not be interpreted as such. Modifications may be made within the spirit and scope of the claims. will be recognized by those skilled in the art.

Impaired leukocyte function in patients with burns covering 20-70% of body surface area The effect of GM-C3F on enhancement can be determined by the following test protocol. : Age 18 years or older, with burns covering 20-40% of the body surface area, cardiovascular Injury to patients who have received or are undergoing stabilization measures and who have no inhalation injury to the lungs. within 48 hours of treatment with recombinant human rhGM-C3F. followed by age 18 years old With burns covering 40-70% of the body surface area, no cardiovascular stabilization treatment was given. E within 48 hours of injury, with no inhalation injury to the lungs. Treat with rhGM-C3F. Next is age 18 years or older, 40-70% of body surface area. with extensive burns, cardiovascular stabilization has been or is ongoing, and with mild to moderate inhalation injury (diagnosed by physical examination or xenon scan) ), patients without bronchoscopy evidence of inhalation were treated with rhG within 48 hours of injury. Treat with M-C3F. rhGM-CSF Lee et al., Proc, Na t1. Acad, Scj, USA, 82:4360 (1985) and US According to the description of Patent Application No. 111.886 (filed on October 23, 1987) That's what I got. rhGM-C3F is in the form of a lyophilized powder and is available from your doctor or Diluted to 1 ml with sterile water and then added 5 Q ml to this resulting solution by a pharmacist. Prepared for intravenous administration by adding saline. The patient is more Preferably rhGM-C8F at 3.10 or 15 micrograms/kg body weight Intravenously (porse or infusion) in doses over 2-4 hours, most preferably 4 hours. It was administered once a day. GM-CSF at each dose level was tested in 3-5 patient groups. was administered to the group.

Blood samples were taken for in vitro analysis to determine leukocyte function. these Blood samples were analyzed by methods known in the art. 50% or more from baseline Higher leukocyte count increases indicate therapeutic efficacy and clinically significant results .

The results for all doses are shown together in Figures 1-5. Ll in Figure 1-5 ``Normal'' or ``Control'' refers to a normal patient population (i.e., without burn injury). L) "Burn control" refers to results with burn injury and no GM-CSF treatment. 0 patient population. In Figure 2-5, "before" is before treatment (i.e. GM-C SF means no administration, and the numbers “1”, “8” and “15” on the X axis are rhG Means the number of days of treatment with M-C8F.

Figure 1 shows the patient group compared to the historical control group. Figure 3 shows a comparison of the uptake of tritiated thymidine. As shown in Figure 1, after burn injury Tritiated thymidine uptake in a study of 4 patients (N=4) over 10 days A significant increase in monocyte proliferation was observed, as evidenced by a significant increase in . Figure 1 There are two types of controls, and the leftmost bar (N=5) is for intracephalic injuries such as burns. λ normal control without intracident disease The bar in the middle (N = 7, 15 days or more after burn injury) represents the group of patients with actual burn injuries. vinegar.

Figures 2 and 3 depict the human oxidation product nostrum upon PMA stimulation of monocytes. child These figures show that continuous administration of rhGM-CSF for over 15 days reduces the acidity of monocytes. This is to prove that the chemical burst increases.

Figures 4 and 5 demonstrate FMLP stimulation of oxidative respiration in humans. These figures are constant FMLP alone did not significantly stimulate monocyte oxidative NOx over a period of time). prove that.

The present invention has been described above, but it will be obvious that the same may be modified in many ways.

It is believed that these changes do not depart from the spirit and scope of the invention; All such modifications are intended to be included within the scope of the claims.

% χ　　　If the control pressure is 0, please tell me. 7=　　　　　　≠Ya Kenyo Tsui Via♀Relie F dimension and say %　　Nigosho (Niteng Ding Mia F (Basian Katsu) Procedural amendment Director General of the Patent Office Wataru Fukasawa 1, Display of the case PCT/US90100379 2. Name of the invention Treatment of leukocyte dysfunction using GM”-C5F 3. Person performing correction Relationship to the case Patent applicant address Name: Schering Corporation 4, Agent Address: 2-2-1-5 Otemachi, Chiyoda-ku, Tokyo, subject to amendment The scope of the claims Special table Hei 3-504980 (5) (Attachment) (1) The scope of claims is amended as follows.

rl, 0ifi Method for treating leukocyte dysfunction associated with physical injury in mammals administering to the mammal an amount of GM-C8F effective to enhance leukocyte function; The method of treatment comprises administering. "that's all international search report international search report

Claims (10)

[Claims] 1. A method for treating leukocyte dysfunction associated with physical injury in a mammal, the method comprising: Administering to a mammal an amount of GM-CSF effective to enhance leukocyte function. The treatment method described above. 2. Administer GM-CSF at a dose of about 3 to about 30 micrograms/kg body weight per dose. The method of treatment according to claim 1, which comprises administering. 3. Claims 1 and 5, wherein the GM-CSF is recombinant human rhGM-CSF. or the treatment method described in Section 2. 4. Claims 1 to 3, wherein the administration is by intravenous infusion or injection. The treatment method described in any of paragraphs. 5. Any of claims 1 to 4, wherein the administration is over a period of about 4 hours. The treatment method described in 6. Claim 1, wherein the dose is about 3 to about 15 micrograms/kg body weight. The treatment method according to any one of Item 5. 7. Claims 1 to 6, wherein the bodily injury is a burn injury. Method of treatment. 8. Leukocyte dysfunction in mammals that accompanies physical injury in mammals. A method of treatment by administering an amount of GM-CSF effective to enhance function Use of GM-CSF for the manufacture of drugs used in. 9. GM-CS for the treatment of patients with leukocyte dysfunction associated with physical injury Use of F. 10. GM used to treat patients with leukocyte dysfunction associated with physical injury - A pharmaceutical composition comprising CSF.